Agent and method for controlling angiogenesis

ABSTRACT

A metal ion chelating agent such as picolinic acid or derivatives thereof, and methods of using the same. The agents chelate metals in metal containing protein complexes and enzymes required for growth and replication of blood vessel cells. The preparations can be administered systemically or for topical use. The preparations have antineoplastic activity augmented by the antiangiogenic properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application Ser. No.60/182,608, filed Feb. 15, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

BACKGROUND OF THE INVENTION

The invention relates to the treatment of diseases and conditionsexacerbated by the development of blood vessels and concomitant bloodflow. More specifically the invention relates to the use of metalchelating materials including, picolinic acid, fusaric acid and theirderivatives as pharmacological response modifiers to reduce angogenesisin the presence of cancers, tumors and in the eye.

It will be appreciated that hereinafter the use of the term “responsemodifier” is intended to encompass all of the intended functions of theinvention and method including antiviral, antiinfective,antiinflammatory, anticancer, vaccine and so on. Further, it will beappreciated that the broad term “antiinfective” is intended to includeantibacterial, antifungal, antiparasitic functions, as well as actionsagainst any other infective agent or organism including viruses notencompassed by the term “antiviral”. It will also be appreciated thatthe term “antiinflammatory” is intended to include an inflammatoryresponse modifier, including all inflammatory responses such asproduction of stress proteins, white blood cell infiltration, fever,pain, swelling and so forth. Furthermore, the term “analgesic” isintended to include a pain reliever, whether the pain incurred is aresult of disease, inflammation, trauma or psychosomatic reaction.

Researchers recently have come to appreciate the role of metalcontaining proteins in physiological actions and responses includingpain, inflammation, proliferative and infectious diseases. Generallyspeaking, the inventor has studied the important function of proteinshaving amino acid sequences which bind metals, particularly transitionmetal ions therein. For example, the inventor has determined theimportant role zinc finger or zinc ring proteins as hormone-receptorproteins and in proliferative, inflammatory and infectious diseases.Moreover, the inventor has determined the role of other metal ioncontaining protein complexes, such as the role of iron finger proteinssuch as iron-finger hormone-receptor proteins in aging andcarcinogenesis.

Likewise, researchers have come to understand the role of certainelements, particularly copper, zinc and iron in angiogenesis.Angiogenesis is implicated in certain diseases states such as cancer anddiabetic retinopathy. Zinc, iron and copper are cofactors inangiogenesis.

The inventor and others have recognized at least three efficientapproaches to inhibiting zinc finger proteins: 1) disruption of the zincfinger by modification of the cysteines which are at least one of thefour binding sites for Zn²⁺ in the zinc finger protein which results inthe ejection of zinc ion; 2) removal of the zinc from the zinc fingermoiety by specific chelating agents; and 3) specific chelating agentsthat form a ternary complex at the site of zinc binding on zinc fingerproteins, resulting in inhibition of the DNA or RNA binding activity ofzinc finger proteins.

Papilloma virus infection results in a number of proliferative diseasesin subjects including warts induced by type 4 human papilloma virus(common warts). Moreover, papilloma virus can cause plantar ulcers aswell as plantar warts. Human papilloma virus infection of the uterinecervix is the most common of all sexually transmitted diseases. Commonlyknown as genital warts, this wide spread virus infection is a seriousdisease that potentially can develop into cervical cancer. Since thevirus is permanently present in cells, infection recurs in a significantpercentage of patients. In many instances, conization of the uterinecervix is required to remove the infected tissue.

Condylomata acuminata, also denoted genital warts, are benign epithelialgrowths that occur in the genital and perianal areas and caused by anumber of human papilloma viruses (HPV) including types 6, 11 and 54.These are low risk viruses which rarely progress to malignancy. However,high risk viruses such as HPV-16 and HPV-18 are associated with cervicalintraepithelial cancer.

The actions of HPV are mediated by specific viral-encoded proteins whichinteract and/or modulate cellular DNA and proteins to produce abnormalgrowth and differentiation of cells. Two proteins of the HPV viralgenome, E6 and E7, are well conserved among anogenital HPVs and both maycontribute to the uncontrolled proliferation of basal cellscharacteristics of the lesions. The E7 oncoprotein is a multi-functionalprotein with transcriptional modulatory and cellular transformingproperties. The E7 oncoprotein is denoted as a “zinc finger” proteinbecause it posesses a sequence motif that is implicated in zinc binding.A strong correlation between zinc binding and the transactivationactivity of E7 has been documented. The HPV-16 E6 protein is a “zincfinger” protein that binds DNA and may have transcriptional propertiessuch that its function may be dependent upon the formation of zincfingers. E6 protein can complex with the cellular tumor suppressorprotein p53 and it is necessary with E7 protein for the immortalizationof primary human squamous cells. Only two proteins of HPV areconsistently expressed and integrated in keratinocytes, the E6 and E7zinc finger proteins. The E6 and E7 proteins are responsible forcontinuous cell proliferation. About twenty HPVs are associated withano-genital lesions and all transformed keratinocytes of these lesionscontain E6 and E7 zinc finger proteins. The E6 and E7 regulate growthand transformation by interfering with cellular p53 and pRb proteins,respectively. Thus, one should be able to control or cure HPV byinactivating E6 and E7, the critical zinc finger proteins which arerequired for replication. When replication of the virus is halted,apoptosis of the virally-infected cells must occur. Thus, one can alterthe epidemiology of, for example, carcinoma of the uterine cervix byinterfering with the function of zinc finger or zinc ring proteins.

Herpes viruses, for example, Herpes Simplex Virus (HSV), has twoimportant viral metalloproteins, a zinc finger protein andribonucleotide reductase, an iron-containing enzyme, which are necessaryfor replication and propagation of the viruses. One can alter the courseof herpes diseases, such as “fever blisters” and genital herpes, byinhibiting the two viral metalloproteins.

The human immunodeficiency virus (HIV) encodes several regulatoryproteins that are not found in other retroviruses. The tat protein,which is one of these proteins, trans-activates genes that are expressedfrom the HIV long terminal repeat and tat is essential for viralreplication. The tat protein of the HIV-1 is a zinc finger protein thatwhen added to certain cells in tissue culture, specifically promotesgrowth. It has been shown that the tat protein of HIV-1 stimulatesgrowth of cells derived from Kaposi's sarcoma lesions of AIDS patients.Other experiments raised the possibility that tat might act as a viralgrowth factor to stimulate replication in latently infected cells oralter expression of cellular genes.

The nucleocapsid p7 protein of HIV has been targeted by the inventor fortreatment of HIV viral infections. The p7 protein is required for thecorrect assembly of newly formed virus particles during the viral lifecycle. Moreover, the p7 protein contains two zinc fingers that arerequired for the recognition and packaging of the viral RNA. Because thezinc finger domain is essential for nucleic acid binding, p7 resistantmutants are highly unlikely to occur. Thus, agents that effectivelyattack the two zinc finger domains of the HIV virus nucleocapsid p7 invivo will decrease the overall number of viral particles that bud offand exit the cells to infect other cells.

The hepatitis C virus is not integrated with DNA and thus may bevulnerable to attack by specific antivirals. The hepatitis C viruses aredependent upon the Zn²⁺ metalloproteinases for specific viral functions.Processing at the C terminus portion of the NS2 protein of hepatitis Cvirus is mediated by virus encoded protease (metalloproteinases).Modification of the metalloproteinases presents an opportunity forcontrolling the progression of hepatitis C mediated disease.

It is of interest to note that the breast cancer susceptibility geneBRCA1 includes a zinc ring domain that is involved in protein-proteininteractions or protein-DNA interactions. It also is of interest to notethat the zinc ring domain of the BRCA1 has a 54% sequence similarity and38% sequence identity with a zinc ring domain encoded by the genome ofthe equine herpes virus. (R. Bienstock, “Molecular Modeling of ProteinsStructures, Science & Medicine, January/February 1997, p.56).

From the foregoing it appears that it would be beneficial to have aproduct that can interfere with the formation or action of certain zincfinger proteins or zinc ring proteins to stop the progress of certainvirally induced or mediated proliferative diseases or to halt theprogress of viruses or malignancies dependent upon zinc finger or zincring proteins for their transformation and immortalization. Furthermore,it would be beneficial to provide a product that can halt the growth ofother proliferative cells, such as malignant cells by chelating metalions from zinc-dependent or iron-dependant, transition metal ion (e.g.copper, iron, etc.) dependent proteins, hormones and enzymes necessaryfor the replication of the malignant cells. Likewise, it would bebeneficial to retard angiogenesis in certain disease states, such ascancers and diabetic retinopathy and post-ophthalmic surgery.

BRIEF SUMMARY OF THE INVENTION

It is among the general objects of the present invention to provide apharmacological response modifier which can retard the growth andproliferation of target cells by blocking the activity of metalion-containing proteins and enzymes.

Another object of the present invention is to provide a compound whichcan retard the growth and proliferation of cells by blocking theactivity of transition metal ion-containing protein structures such aszinc finger, zinc ring proteins, iron-finger or iron-ring proteins orenzymes associated with replication of the cells.

Another object of the invention is to provide such a compound to retardangiogenesis, particularly angiogenesis in tumors and in the eyefollowing ophthalmologic surgery.

Yet another object of the invention is to provide such a compound thatinhibits fibroblast production after cataract surgery which results froman inflammatory response to artificial lenses.

Another object of the invention to provide a method of halting thefunction of metal containing protein structures containing metals otherthan zinc, metal-containing ring proteins structures, such asiron-finger or iron-ring proteins and other proteins with metal bindingmotifs heretofore unidentified by the administration of a metalchelating agent, both topically and systemically.

Yet another object of the invention is to provide such chelating agentsin a relatively safe and nontoxic form such as picolinic acid or itsderivatives for both topical and systemic use.

According to the invention, briefly stated, a method or treatment andcompound used in the method, for example, metal chelating compounds,such as picolinic acid or derivative thereof, for the treatment ofdiseases which have, as a mode of progression or as a symptom orsequelae, angiogenesis or enhanced angiogenesis, as well as the methodof treatment. The invention can be used orally or topically to treat orcontrol a number of diseases. The metal chelating compounds bind metal,for example iron or transition metal ions such as zinc or copperrequired by cells for angiogenesis. In one preferred embodiment, an oraldose of 500 mg of picolinic acid is administered over a five weekescalation from approximately 500 mg daily to approximately four (4)grams daily, in divided doses.

Various active derivatives that maintain their activity and stabilitywhen after systemic administration are provided. Slow release oralformulations can be used to treat diseases for the digestive tract. Theactive derivatives can be administered orally, parenterally, byinhalation, transdermally, intrathecally, implantation under the skin orby any other appropriate method to control proliferation or bloodvessels in the disease state.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, FIG. 1 is the chemical structure of fusaric acid;

FIG. 2 illustrates the effect of picolinic acid on total protein of LoVocells;

FIG. 3A illustrates the effects of fusaric acid on the growth of WI-38cells;

FIG. 3B illustrates the effects of fusaric acid on the growth LoVocells;

FIG. 3C illustrates the effects of fusaric acid on the growth of KBcells;

FIG. 4A illustrates the effects of fusaric acid on morphology of WI-38cells, the cells treated without fusaric acid;

FIG. 4B illustrates the effects of fusaric acid on morphology of WI-38cells, the cells treated with fusaric acid;

FIG. 4C illustrates the effects of fusaric acid on morphology of LoVocells, the cells treated without fusaric acid;

FIG. 4D illustrates the effects of fusaric acid on morphology of LoVocells, the cells treated with fusaric acid;

FIG. 5A illustrates the effects of fusaric acid on morphology of KBcells, the cells treated without fusaric acid;

FIG. 5B illustrates the effects of fusaric acid on morphology of KBcells, the cells treated with fusaric acid;

FIG. 6 illustrates modulation of apoptosis by intracellularconcentrations of zinc;

FIG. 7 illustrates the role of zinc finger proteins in HIV infection;

FIG. 8 illustrates the effect of picolinic acid and derivatives on HIV,zinc finger proteins and retroviruses;

FIG. 9 illustrates disruption of zinc finger binding domains inretroviral proteins caused by picolinic acid;

FIG. 10 illustrates the wide spectrum of antiviral activity of picolinicacid;

FIG. 11 illustrates the molecular structure of a derivative of picolinicacid for system use;

FIG. 12 illustrates the binding of a picolinic acid derivative to zincand adjacent amino acids of a zinc finger protein; and

FIG. 13 is a schematic illustrating the interrelation of viralinfection, ribosomal proteins and the zinc finger heat shock protein,DnaJ which is involved in inflammation.

DETAILED DESCRIPTION OF THE INVENTION

Picolinic acid, a metal chelating, naturally occurring, biologicalcompound, which inhibits the growth of numerous cultured normal andtransformed mammalian cells. Picolinic acid has the chemical name of2-Pyridine carboxylic acid, also known as alpha-pyridine carboxylicacid, having the chemical formula C₆H₅NO₂, molecular weight: 123.11g/mol, and is readily soluble in water. Picolinic acid has an LD-50 of140 grams in a 70 kg subject.

It has been shown that short-term treatment with picolinic acid arrestsnormal cells in G₁, (Go) while transformed cells are blocked indifferent phases of the cell cycle. With longer exposure to picolinicacid cytotoxicity and cell death was observed in all transformed cellswhether they were blocked in G₁, G₂ or at random. In contrast, mostnormal cells showed no toxic effects from the picolinic acid. Thus, theselective growth arrest and the differential cytotoxicity induced bypicolinic acid reveals a basic difference in growth control and survivalmechanism(s) between normal and transformed cells.

Kinetic and radioisotopic studies show that picolinic acid both inhibitsincorporation of iron into the cells and effectively removes radioironfrom the cells. Hence, it is conceivable that the inhibition of cellproliferation in vitro, as well as tumor growth in vivo, by picolinicacid results, at least in part, from selective depletion of iron in thecells.

However, it also is shown that picolinic acid may arrest prokaryote andeukaryote cell growth by inhibiting Zn-requiring enzymes. In addition toits chelating ability, picolinic acid has a number of biologicproperties such as inhibitory effects on ADP ribosylation and ribosomalRNA maturation, modulation of hormonal responses, and macrophageactivation. Picolinic acid in combination with interferon-γ can inhibitretroviral J2 mRNA expression and growth in murine macrophages. Thus,picolinic acid and its derivatives can act as a biological responsemodifier.

The inventor has determined that picolinic acid and fusaric acid werefound to inhibit the zinc dependent binding of recombinant MPS-1 to DNA,as determined by gel shift assays and the data correlates with theabsence of radioactive ⁶⁵Zn from recombinant MPS-1 protein. MPS-1 is aubiquitous tumor marker and cell growth stimulator and is described indetail in the inventor's U.S. Pat. No. Re.35,585 (No. 5,243,041). MPS-1has one zinc finger domain of the type CCCC. Picolinic acid and fusaricacid react with the CCCC zinc finger to remove radioactive ⁶⁵Zn fromMPS-1. This is detected by a change in the electrophoretic mobility ofMPS-1 under non-denaturing conditions. These experiments indicate thatpicolinic acid and derivatives should remove zinc and denature varioustypes of zinc finger or zinc ring proteins, whether known or heretoforeundiscovered, including viral proteins such as nucleocapsid p7 proteins,as will be explained further. Furthermore, the inventor has determinedthat any chemical compound, whether known or heretofore undiscovered,that will remove the zinc (or other metal) and denature the proteins orthat will form a ternary complex (protein-zinc-chelator) can beeffective as a therapeutic agent or as an autologous immune responsemodulator, as will be discussed in greater detail below.

Fusaric acid is a potent inhibitor of cancerous cell growth. Fusaricacid, a picolinic acid derivative, metal ion chelator, shows an effecton the growth and viability of normal and cancerous cells in tissueculture. Examples presented here show that fusaric acid has potentanti-cancer and anti-viral activity in vitro. Moreover, fusaric acid maybe useful in the treatment of spontaneous and virally-induced tumors invivo without substantially damaging living normal cells.

Fusaric acid is the 5-butyl derivative of picolinic acid. Its structureis shown in FIG. 1. Fusaric acid was recognized in the early 1960's tohave activity as an antihypertensive agent in vivo. Fusaric acid and itsproperties can be summarized as follows. Undoubtedly the drug interactswith various metalloproteins and metal ion-requiring enzyme systems.Fusaric acid is noted to be an inhibitor of a wide variety of seeminglyunrelated enzyme systems. These include poly ADP ribose polymerase, aZn-finger enzyme, and other Zn-finger proteins. Cu-requiring systems arealso effected by fusaric acid. These enzymatic systems are important ingrowth control mechanisms. It has become increasingly clear that fusaricacid, by virtue of its butyl group penetrates the cell interior muchmore easily than picolinic acid, and works at least in part as a Zn/Cuchelating agent.

As mentioned above, the hepatitis C family of viruses are dependent uponmetalloproteinases having a zinc finger domain for replication of thevirus. Picolinic acid, fusaric acid or other suitable derivatives oranalogs, can be administered orally to patients exposed to or sufferingfrom hepatitis C related disease to bind the metal in themetalloproteinases and thereby control the disease. Furthermore, theoral administration of the metal chelator in combination withinterferons will result in the elimination of the virus from the cellsbecause the hepatitis C virus is not integrated with the DNA and thus isvulnerable to this double attack.

Novel substituted derivatives of picolinic acid and related compoundscan be used systemically to treat cancer, viral infections and otherrelated diseases and proliferative disorders and undesired angiogenesis.The novel substituted derivatives of picolinic acid and relatedcompounds also work by disrupting the binding of zinc atoms in zincfinger proteins, zinc ring proteins or other structures heretoforeunknown that depend upon the inclusion of zinc or other metal ions suchas transition metal ions, for stability, packaging or replication.Further, the novel substituted derivatives are stable and retain theirzinc chelating properties even when introduced systemically byinjection, oral administration, inhalation or transdermal or otherroutes of administration.

FIG. 11 illustrates novel derivatives of picolinic acid for systemicuse. Computer modeling indicates that such derivatives can interact withzinc atoms and disrupt their binding to the zinc finger protein.Substitutions at positions 3, 4, 5 and 6 on the 2-pyridine carboxylicacid (picolinic acid) have the proper configuration to preventinterference with the zinc finger protein backbone. For example R1, R2,R3 or R4 can be a methyl, ethyl, propyl, isopropyl, butyl, isobutyl,secondary butyl, tertiary butyl, pentyl, isopentyl, neopentyl or similargroup. Further, substitution with halogens such as fluorine, chlorine,bromine and iodine can result in effective, systemically active agents.The systemic compounds can be prepared by methods generally known to theart and include pharmacologically acceptable salts thereof.

FIG. 12 illustrates the binding of zinc in a zinc finger or zinc ringprotein by derivatives of picolinic acid. Further, as shown in FIG. 12,the substituted positions at positions 3, 5 or 6 i.e. R1, R3 or R4 canattach to amino acids on each side of the zinc thus binding the zinccontaining protein at three sites and forming ternary complex comprisedof the protein, the zinc, and the picolinic acid derivative whichinactivates the protein. Therefore, the above-listed moieties that canbe substituted at the various positions can result in a picolinic acidderivative that not only is more stable for systemic administration, butalso one that has even greater affinity and specificity for, and bindingpotential with, various zinc finger or zinc ring proteins.

It will be appreciated that substitutions at the 3, 4, 5 and 6 positionscan be made with a peptide of sixteen amino acids or more with eitherbasic or acid amino acids predominating. The substituted picolinic acidwould have an increased molecular weight and a substantially increasedhalf-life in the blood. Further, such compounds would penetrate thevirus-containing cells more effectively due to the amphipathic nature ofthe peptide residues.

The systemic compounds can be administered to human and animal subjectsby any means that produces contact of the active agent with the targetprotein, such as orally, parenterally, inhalation, transdermally,rectally, intrathecally, implanted under skin on any other method forobtaining a pharmacologically acceptable blood level. In general, apharmacologically effective daily does can be from about 0.01 mg/kg toabout 25 mg/kg per day or any other pharmacologically acceptable dosing.In general, a pharmacologically effective daily does can be from about0.01 mg/kg to about 25 mg/kg per day or any other pharmacologicallyacceptable dosing. A pharmaceutically active and acceptable preparationof picolinic acid or derivative in a concentration of approximately 1%to approximately 99%, preferably in a daily range of approximately 250mgto 6000 mg, preferably approximately 500 mg to approximately 2000 mg ofpicolinic can be used for this mode of treatment. It will be appreciatedthat doses approximating the LD-50 of 140 grams/70 Kg may be covered bythe invention in the event continued research shows higher doses areoptimal.

It will be appreciated that picolinic acid derivatives referred toherein as the systemic compounds can be employed in the hereinafterdescribed topical preparations as well as employed systemically.Furthermore, the claimed invention is intended to include any otherchemical compounds, either derivatives of picolinic acid, compounds withstructural relationships to picolinic acid, or heretofore unknowncompounds that function to chelate, attach to, or modify metal ions inproteins structures, including, but not limited to transition metal ionsfound in proteins structures of viruses, proliferative cells (plant oranimal) or even as components of fungi and bacteria.

It previously has been discovered that p7 protein is required forcorrect assembly of newly formed virus particles during the viral lifecycle, as explained above. By modeling, the inventor has discovered theactivity of picolinic acid in disrupting zinc finger proteins inretroviruses, as is illustrated in FIGS. 6-10. FIG. 6 illustratesmodulation of apoptosis by intracellular concentrations of zinc; FIG. 7illustrates the role of zinc finger proteins in HIV infection; FIG. 8illustrates the effect of picolinic acid and derivatives on HIV, zincfinger proteins and retroviruses; FIG. 9 illustrates disruption of zincfinger binding domains in retroviral proteins caused by picolinic acid;and FIG. 10 illustrates the wide spectrum of antiviral activity ofpicolinic acid.

The p7 protein contains two zinc fingers that are required for therecognition and packaging of viral RNA. In one embodiment and aspect ofthe invention, the inventor has targeted p7 for drug therapy withpicolinic acid and derivatives. Picolinic acid and derivatives are zincfinger disrupting agents that act by attacking the two zinc fingerdomains of the virus (i.e. HIV) nucleocapsid p7 in vitro. This resultsin picolinic acid and derivatives inducing an overall decrease in thenumber of viral particles that bud off and exit the cells to infectother cells. It is known that HIV-1 contains two zinc fingers in theretroviral p7 protein. The zinc fingers are highly conserved throughoutessentially all retroviruses. Thus, mutations in the zinc fingers of theHIV-1 virus will produce a non-infectious HIV-1 particle. Because thezinc finger domain is essential for nucleic acid binding, p7 resistantmutants will not occur. The picolinic acid can be used, therefore, forprevention of retroviral and other viral diseases by, for example,inhibiting exit of the virus or virus particles from the cells or bychemically inducing a non-infectious virus. Furthermore, any chemicalentity, either known or unknown at this time, that functions in the samemanner as picolinic acid or its derivatives, is intended to beencompassed by the instant invention. Representative viruses whichinclude zinc finger or zinc ring proteins are included on Table 1.

TABLE 1 Examples Of Families Of Viruses Using Zinc Finger Proteins, ZincRing Proteins Or Transition Metal Ion-Dependent Enzymes For ReplicationAnd/Or Virulence Location and general Protein Function and Virus proteinand Mr Characteristics Specific Properties Reovirus Lambda-1, 140 KdInner capsid Zinc finger protein Binds dsDNA Rho-3, 41 Kd Outer capsidZinc finger protein Binds dsRNA Rotavirus NSP1, 53 Kd Non-structuralZinc finger protein RNA binding Retroviridae Ncp7 (AIDS) NucleocapsidZinc finger protein 55 amino acids RNA binding Required for inclusion ofRNA in virions TAT (AIDS) Regulatory protein Cluster of 7 cysteineresidues 82-101 amino acids Trans-activator Papillomavirus E6 Regulatoryprotein Zinc finger protein Transforming protein of HPVs Continuous cellproliferation Targets degradation of p53 E7 Regulatory protein Zincfinger protein Transforming protein of HPVs Continuous cellproliferation Binds to the retinoblastoma protein, Rb Adenovirus E1ARegulatory protein Zinc finger protein Gene expression Transformingprotein Hepatitis C NS2(+NS3) Zn-dependent enzyme Zn-metalloproteinaseHerpes viruses HSV-1: ICPO protein Regulatory protein Zinc fingerDNA-binding Trans-activation HSV-2: MDBP protein Regulatory protein Zincfinger protein ssDNA-binding DNA replication ICP6: RibonucleotideFe-dependent Synthesis of DNA precursors Reductase Enzyme Equine Herpesvirus-1 Regulatory protein Zinc ring configuration ZR protein DNAbinding Protein/protein interactions

The chelating agents of the present invention can be used aspreservatives in perishable items such as foods and pharmaceuticals andto prevent fungal growth on the surface of fresh fruits. Presentlychemicals such as citrashine orthophenilphenol thiabendazole are used.Stability experiments have shown that picolinic acid, for example, ishighly stable when used as a preservative in foods and on the surface offresh fruits. Microbial and fungal growth is inhibited while the foodcomponents are unaffected. The preservative of the present invention hasless likelihood of toxicity or untoward reactions if ingested that thepresent, complex chemical antifungals. The preferred concentration ofpicolinic acid or acceptable derivative for this use is approximately 1%to approximately 99%.

The chelating agents of the present invention are used to controlangiogenensis. New blood vessels are formed because copper Cu²⁺ isavailable to stimulate certain enzymes. Angiogenesis can be problematicin two specific situations. First, the increased blood vessel formationin tumors and the increased blood vessel formation in the eye,particularly after ophthalmologic surgery. Increased angiogenesis, incombination with increased fibroblast production, as will be discussedin greater detail below, can result in opacity of the ocular lens. Theadministration of the novel chelating agent, particularly picolinicacid, fusaric acid or acceptable derivatives or analogs, preventunwanted angiogenesis. In the case of tumor control, the chelating agentcan be administered orally or injected directly into the tumor. In thetreatment of the eye, the product can be administered orally orpreferably, topically to the eye.

Examples of the specific effects of metal chelating agents, includingpicolinic acid, substituted picolinic acid derivatives and fusaric acid,as well as the practical application of those agents will now bedescribed:

EXAMPLE 1 Effects of Picolinic Acid on Growth of WI-38 LoVo and KB,Cells

Cells were plated at 1.5×10⁵ cells/60-mm dish; 48 hours later, themedium was removed, and new media with or without 3 mM picolinic acidwas added. Total cell protein was determined at the indicated times;each point is the average of triplicate measurements from 2 cultures.

The growth of normal WI-38 cells was inhibited by 3 mM picolinic acidwithin 24 hours, the cells showed no toxic effects for up to 72 hours oftreatment, and the inhibition was reversible within 24 hours of removalof the agent (data not shown). These results are identical to previousresults with WI-38 cells incubated with picolinic acid.

The growth of LoVo cells was inhibited by 3 mM picolinic acid (FIG. 2).After 24 to 48 hours of exposure to picolinic acid (3 mM), LoVo cellsacquired a flattened morphology, they began to look granular, no mitosiswere observed, and some began to float in the medium, (data not shown).With longer exposure (48-72 hours) cytotoxicity and cell death wasobserved in LoVo cells (data not shown). Equivalent results wereobtained with cancerous KB cells treated with picolinic acid (3 mM) butits cytotoxic effects on this cell type were not as pronounced as in thecase of LoVo cells (data not shown).

EXAMPLE 2 Effect of Fusaric Acid on Growth and Viability of Normal WI-38Cells

In initial experiments to examine the effects of fusaric acid on cellgrowth and viability, WI-38 and LoVo cells were incubated for 24 to 72hours in medium with or without various doses of fusaric acid (0.1-1mM). The growth of both WI-38 and LoVo cells was inhibited by 500 μMfusaric acid in a time and dose dependent manner, as shown below inTable 2. A higher dose of fusaric acid (1 mM), caused a pronounceddecrease in the rate of cell growth of both cell lines, and extensivecytotoxicity was noted particularly in LoVo cells by 24 hours. Thesepreliminary experiments led to detailed tests of the effects of thehighest dose of fusaric acid (500 μM) which appeared to show somedifferential toxicity on LoVo cells with little toxicity to WI-38 cells(Table 2).

TABLE 2 Effect of Different Doses of Fusaric Acid on WI-38 and LoVoTotal Cell Protein Monolayer Protein (μg/dish)^(a) Addition 0 h 24 h 48h 72 h WI-38 None 105 202 270 371 Fusaric acid (0.5 mM) — 195 275 345Fusaric acid (1 mM) — 236 202 195 LoVo None 202 270 352 457 Fusaric acid(0.5 mM) — 135  90 101 Fusaric acid (1 mM) — ND ND ND ^(a)Cells wereplated at 1.5 × 10⁵ cells/60-mm dish in DME/F12 medium containing 10%Calf serum. The medium was removed 24 hours later and then fresh mediacontaining the indicated concentrations of fusaric acid were added.Protein was determined at the indicated times. Points are the mean ofduplicate determinations. SE did not exceed 5% of the mean. ND, not donebecause of extensive cell destruction.

FIG. 3A shows that the growth of WI-38 cells was strongly inhibited by500 μM fusaric acid. After 30 to 48 hours in 500 μM fusaric acid, WI-38cells acquired a more flattened morphology, showed some granularity, andno mitotic cells, as illustrated in FIG. 3B, or further increase in cellnumber were observed (See, FIG. 3A). Following 30 hours incubation withfusaric acid (500 μM), normal growth rate was not restored after removalof fusaric acid and the cell number decreased significantly (30%) after4 days in normal media. The remaining cells were spread on thesubstratum in normal manner without any visible mitosis for 4 days afterremoval of the drug. However, they resumed growth after 125 hours ofremoval of fusaric acid (FIG. 3A), and most (>95%) of the cellssurvived. These results suggest that the majority of WI-38 cells werearrested in G₁(G₀) by fusaric acid and they proceeded slowly through thecell cycle after its removal.

To examine WI-38 cell viability in greater detail, the effects offusaric acid were studied in logarithmically growing and contactinhibited confluent cells (Tables 2 and 3). In logarithmically growingWI-38 cells approximately 76% of the cells were viable after 30 hours oftreatment with fusaric acid. When the cells were treated for 78 hours,only 26% of the cell population survived the pronounced cytotoxicactions of fusaric acid. The data are shown below in Table 3.

TABLE 3 Viability of Cells in Logarithmic Growth After Treatment withFusaric Acid^(a) % Survival^(b) Cell line 30 h 78 h WI-38 Control 100100 Treated 76.4 26 LoVo Control 100 100 Treated 38.5 4.5 ^(a)The cellswere incubated in medium with or without 500 μM fusaric acid for theindicated times. ^(b)Fraction of total cells counted which did not stainwith trypan blue. Cells attached to the dish were exposed to trypan blueand counted. The percentage exclusion by untreated cultures wasnormalized to 100% for comparison with fusaric acid-treated cultures.

The detached cells showed conspicuous cytotoxic effects and most of themwere destroyed. Interestingly, in confluent cells, fusaric acid did notshow any cytotoxic effects as determined by the fact that 100% of thecells survived 48 hours of treatment with 500 μM fusaric acid, as shownin Table 4, below.

TABLE 4 Viability of Confluent Cells after Treatment with Fusaric Acid(500 μM) % Survival^(a) Cell line Control Treated WI-38 100 100 LoVo 100 40 KB 100  95 ^(a)Determined at 48 h using trypan blue dye exclusiontest as indicated in Table 3.

Thus, a significant proportion of the population of growing cells (76%)and all of the confluent WI-38 cells cell resisted the marked cytotoxicaction of fusaric acid.

EXAMPLE 3 Effect of Fusaric Acid on Growth and Viability of ColonCarcinoma LoVo Cells

Fusaric acid (500 μM) inhibited LoVo cell growth, as shown in FIG. 3B.After 30 hours of treatment with 500 μM fusaric acid, there was aprominent decrease in cell number. DNA synthesis was completely (100%)inhibited by 24 hours. When treated with 500 μM fusaric acid, themajority of the LoVo cells acquired a rounded morphology by 48 hours.

As shown in FIG. 4D, most of the cells became granular, showedpronounced cytotoxic effects, many were destroyed, and subsequentlydetached from the culture dish. These floating cells were not viable.They did not adhere to the substratum and disintegrated after 1 to 3days when resuspended in fresh medium without fusaric acid. FIG. 4Bshows that within 30 hours of treatment there was a 60% decrease in cellnumber. Following removal of the drug after 30 hours of treatment showedthat the cell population continued to decline (˜80%) in number up toapproximately 100 hours (FIG. 4B). However, after 100 hours, an increasein cell number was noted after 25 additional hours.

As in the case of WI-38, LoVo cell viability after fusaric acidtreatment was investigated in logarithmically growing and confluentcells, as shown in Tables 2 and 3. In logarithmically growing LoVocells, approximately 38% of the attached cells were viable after 30hours of treatment with fusaric acid. When the cells were treated for 78hours, only 4.5% of the cell population survived the pronouncedcytotoxic actions of fusaric acid. The detached cells showed noticeablecytotoxic and most of them were destroyed at these time points. Inconfluent cells, fusaric acid showed a significant cytotoxic effect asdetermined by the fact that only 40% of the cells survived 48 hours oftreatment with 500 μM fusaric acid. Thus, LoVo cells are much moresensitive to the cytotoxic actions of fusaric acid in both growing andconfluent population of cells in comparison to normal WI-38 cells.

EXAMPLE 4 Effect of Fusaric Acid on Growth and Viability of HumanCarcinoma KB Cells

FIG. 2C shows that the growth of KB cells was inhibited by fusaric acid(500 μM). After 24 hours of treatment there was no further increase incell number. As illustrated in FIGS. 3C and 5B, after 24-48 hours in 500μM fusaric acid, most of the KB cells acquired a more flattenedmorphology, and no mitotic cells or further increase in cell number wereobserved. Following 48 hours incubation with fusaric acid (500 μM),normal growth rate was not restored after removal of fusaric acid andthe cell number decreased significantly after 27 additional hours innormal media (FIG. 3C). The remaining cells were spread on thesubstratum in normal manner without any visible mitosis for 27additional hours after drug removal. However, they resumed growth after27 hours of removal of fusaric acid (FIG. 3C).

To examine KB cell viability in greater detail, the effects of fusaricacid were studied in logarithmically growing and confluent cells. Inlogarithmically growing KB cells 70% of the cells were viable after 48hours in 500 μM fusaric acid. In confluent cells, fusaric acid did notshow significant cytotoxic effect, as determined by the fact that 95% ofthe cells survived 48 hours of treatment with 500 μM fusaric acid (SeeTable 4, above). Thus, in contrast to LoVo cells, a significantproportion of the population of growing (70%) cells and virtually all(95%) of confluent KB cells resisted the pronounced cytotoxic action offusaric acid (See Tables 3 and 4, above).

EXAMPLE 5 Effect of Fusaric Acid on Growth and Viability of Human BreastAdenocarcinoma Cells

Fusaric acid (500 μM) rapidly inhibited human breast adenocarcinomaMDA-468 cell growth. After 12-24 hours of treatment with 500 μM fusaricacid, there was no further increase in cell number. DNA synthesis wascompletely inhibited (100%) by 24 hr. When treated with 500 μM fusaricacid, the majority of the MDA-468 cells became granular, showedpronounced cytotoxic effects, many were destroyed and subsequentlydetached from the culture dish. These floating cells were not viable.Within 30 hours of treatment there was a 65% decrease in cell number.Following removal of the drug after 30 hours of treatment showed thatthe cell population continued to decline in number. After 96 hours, lessthan 10% of the original population remained attached to the dish and nochange in cell number was noted after one additional week.

As in the case of WI-38, MDA-468 cell viability after fusaric acidtreatment was investigated in logarithmically growing and confluentcells. In logarithmically growing MDA-468 cells, less than 20% of theattached cells were viable after 30 hours of treatment with fusaricacid. When the cells were treated for 48 hours, only 0.1% of the cellpopulation survived the pronounced cytotoxic actions of fusaric acid. Inconfluent cells, fusaric acid showed significant cytotoxic effect asdetermined by the fact that only 10% of the cells survived 48 hours oftreatment with 500 μM fusaric acid. Thus, MDA-468 cells are extremelysensitive to the cytotoxic actions of fusaric acid in both growing andconfluent population of cells in comparison to normal WI-38 linesstudied.

Thus, fusaric acid is effective to reduce and control growth of thiscommon type of human malignancy.

EXAMPLE 6 Effects of Fusaric Acid on Growth and Viability of Other HumanCarcinoma Cell Types

As in previous examples, the following human cell lines were inhibitedby similar concentrations of fusaric acid: Prostatic adenocarcinoma,skin carcinoma, colon carcinoma, liver adenocarcinoma and lungadenocarcinoma. For all these cell types, cell viability decreased byapproximately 60% after 48 hours of treatment with fusaric acid.

EXAMPLE 7 Combined Effects of Fusaric Acid and Standard ChemotherapeuticAgents

Other chemotherapeutic agents such as 5-fluorouracil and/or levamisolein the case of colon adenocarcinoma may be utilized in conjunction withfusaric acid to enhance the effectiveness of therapy. Irreversible celldeath and biological alterations induced by fusaric acid also may beenhanced by using agents from the group consisting of anti-cancerantibodies, radioactive isotopes, and chemotherapeutic agents.

The method of using fusaric acid or picolinic acid topically to treat avariety of viral and spontaneous proliferative diseases in human andanimal subjects, as will be described in detail below, can be used incombination with cytotoxic agents selected from the group consisting ofchemotherapeutic agents, antibodies, radioactive isotopes, and cytokines(e.g. Interferons), vitamin A, for enhanced activity.

EXAMPLE 8 Fusaric Acid Effect on Cells with Increased P-protein Activity

Multidrug resistance (MDR) is a formidable obstacle to effective cancerchemotherapy. Studies have indicated that MDR is a phenomenon in whichresistance to one drug is associated with resistance to a variety ofunrelated drugs. Thus, even when a combination of chemotherapeutics isused, patients may exhibit concurrent resistance to some or all of thedrugs, leading ultimately to failure of therapy.

One of the primary contributors to MDR is a glycoprotein denotedP-glycoprotein of molecular weight 170 Kdal, also known as P170.P-glycoprotein or P170 acts as a pump, effectively eliminatingchemotherapeutic agents from the cell interior to the extracellularspace. Although drug-sensitive cells are destroyed during the initialand subsequent courses of chemotherapy, drug resistance cells,containing elevated levels of P-glycoprotein, emerge, multiply andeventually lead to death of the host.

P-glycoprotein, the product of the mdr-1 gene is a plasma membraneprotein. The molecule is composed of 12 transmembrane domains and twobinding sites for ATP, which furnishes the energy required for drugelimination. The function of this protein in normal cells is presumablyto eliminate naturally occurring toxic compounds. Elevated levels ofP-glycoprotein have been associated with multidrug resistance innumerous malignancies, including: colon carcinoma, breast carcinoma,liver, pancreas, lung carcinoma and other tumors.

From the previous information, it is evident that drugs that are notneutralized by the P-glycoprotein mechanism will be of benefit forchemotherapeutic attack of susceptible and MDR-resistant cells. Ofconsiderably interest for this invention is the data showing thatfusaric acid does not induce P170 protein and is effective incontrolling growth of cells with high levels of P170 protein. Thus,fusaric acid may have some role in the treatment of tumors which areresistant to MDR-associated drugs.

EXAMPLE 9 Use of Fusaric Acid to Reduce the Expression of RetroviralmRNA Levels

By using Kirsten (K) sarcoma retrovirus-transformed NRK cells it wasshown in preliminary experiments that fusaric acid reduces theexpression of retroviral mRNA levels. Furthermore, it also may be shownthat the combination of fusaric acid and interferon-γ results in apotent inhibition of K sarcoma virus mRNA expression in K-NRK cells.

Identification of fusaric acid as a substance that can inhibitexpression of mRNA controlled by a retroviral promoter is a greatinterest because of the importance of retroviruses, such as the humanimmunodeficiency virus (HIV), in animal and human disease. Although thebiology of K-virus and HIV is different, fusaric acid may be effectivein controlling HIV viral expression. Furthermore, the combination offusaric acid plus interferon-γ may be much more potent in inhibiting HIVexpression in human monocytes and other infected cells. Thus, thisinvention is not limited to the effects of fusaric acid in K-NRK, cellsbut is extended to the actions of this agent in other retrovirallyinfected human and animal cells.

EXAMPLE 10 Treatment of Ulcerative Lesion with Topical Picolinic Acid

A subject horse had a 3 inch diameter ulcerative lesion on the left sideof its neck. The lesion had a papillomatous appearance with bleeding atthe tips of the papillae. The lesion was progressive, with total loss ofhair over the area. The diagnosis was viral disease, i.e. papillomavirus, complicated by fungal infection. The horse was treated withconventional local antibiotic and chemical therapies for about fourmonths. However, the agents used did not modify the course of thedisease.

An aqueous solution of 10% picolinic acid in deionized water was appliedevery other day with a cotton swab over and around the lesion. Thetreatment continued for 45 days. The course of the regression of theviral lesion was as follows:

1) after 10 days of treatment, the bleeding papillae suffered centralnecrosis and the borders of the ulcer acquired the aspect ofgranulomatous proliferating healing tissue;

2) after 20 days of treatment, the healing lesion began to show hairgrowth in multiple areas; the diameter of the lesion was reduced toapproximately 2 inches and appeared flat and clean of debris;

3) after 30 days of treatment, the lesion was about 1 inch in diameterwith abundant hair growth on the borders and on the surface of thelesion;

4) at 45 days the lesion resolved with some scar tissue; hair coveredall of the area; and

5) after three additional months the horse was observed without evidenceof recurring disease.

EXAMPLE 11 Treatment of Patients with Papilloma Virus Skin Lesions

Picolinic acid and its analogues act by chelating metal ions. In thecase of the inhibition of viral replication by picolinic acid, the ioninvolved is zinc, which is essential to maintain the active structure ofzinc finger proteins such as E6 and E7 proteins of the human papillomaviruses essential for viral replication.

Five patients ranging in age from 11 years to 52 years and each havingat least one common wart induced by human papilloma viruses was treatedwith a topical preparation of picolinic acid. The topical preparationwas either solution of 10% to 20% picolinic acid in deionized water or atopical ointment wherein 10% picolinic acid is incorporated intoAquaphor, i.e.1 g of picolinic acid in 10 g of Aquaphor. After sevendays of application of the solution or ointment, central necrosis of thewart occurred. After approximately 4 to 6 weeks the warts were gone. Itshould be noted that there was no significant difference observed in thecourse of disease between the 10% and 20% solutions. However, fasterresolution was seen with the ointment and is believed to be due to thecontinual contact time imparted by the ointment base.

EXAMPLE 12 Treatment of Virus-induced Plantar Ulcer

A 50 year old patient with recurrent plantar wart of about 2 cm indiameter was treated with topical picolinic acid. The patient, apathologist who had difficulty walking because of the pain caused by theulcer, had experimented with numerous medications for more than threemonths without any significant results prior to treatment with thepicolinic acid. It is relevant to note that many plantar ulcers aretransformed into malignant tumors.

The patient was treated with a solution of 10% picolinic acid indeionized water for one week. Central necrosis was noted. He then wastreated with 10% picolinic acid in Aquaphor. The ointment was placed onthe ulcer and on a patch. The patch was replaced every 24 hours. Afteran additional three weeks the plantar ulcer resolved.

EXAMPLE 13 Treatment of Metastatic Disease to the Skull from BreastCancer

A 73 year old female with metastatic breast cancer to the skin and boneof the skull was treated with a topical preparation of 10% picolinicacid in Aquaphor. The preparation was applied to the cancerous lesionsand to a bandage and changed twice daily. The multiple cancer lesionswere approximately 1 to 1.5 cm in diameter. The lesions resolved withscar tissue forming after approximately 35 days.

EXAMPLE 14 Treatment of Proliferative Skin Disorders

Several patients suffering from proliferative skin disorders such aspsoriasis have been included in a recent ongoing study of theanti-proliferative effects of topical picolinic acid. Preliminaryinformation indicates that the picolinic acid has a significant effectin inducing regression of the psoriasis. The patients may be treatedwith a topical application of approximately 5% to 20% picolinic acid, ora derivative thereof, in an absorption base. Alternatively, the patientmay be treated with a solution containing approximately 5% to 20%picolinic acid, or derivative, in deionized water. The topicalpreparation may be applied twice a day or in an alternativepharmacologically acceptable regimen.

EXAMPLE 15 Treatment of Actinic Lesions

Two patients with actinic lesions (average of 5 lesions per patient,each lesion being approximately 3 mm to 5 mm in diameter) were diagnosedas requiring liquid nitrogen removal of the lesions. The patientsreceived a daily application of 10% picolinic acid in Aquaphor. Afterapproximately three weeks of treatment, the lesions were completelycured (eliminated) without any effects on normal skin.

EXAMPLE 16 Treatment of Herpes

The subject was a 58 year old Caucasian male with at least one “coldsore” or common “fever blister”. The lesion was a typical herpes simplexlesion. The subject has a history of such lesions and has treated themwith lip balm or Blistex® with only limited symptomatic relief. Thesubject applied the subject topical antiviral as a 10% aqueous solution.Within twelve hours of the first application, the subject's lesion beganto shrink with a decrease in soreness and pain. After approximately 24hours from the initial application, the lesion as almost completelyhealed. He made a third application approximately 36 hours after thefirst application. Between 36 hours and 48 hours after the initialapplication, the subject described the fever blister as “gone” and“healed”.

EXAMPLE 17 Treatment of Herpes

The subject was a Caucasian female in her mid-fifties with a longhistory of recurring, painful herpes simplex lesions described as “feverblisters”. The subject presented with a painful lesion on her lip. Sheapplied a 10% aqueous solution of the subject antiviral to the lesionapproximately three or four times at 12 hour intervals. She reportedthat the lesion was nearly gone after the third application, but madethe fourth application to “make sure”.

EXAMPLE 18 Treatment of Herpes

The same female subject of Example 17 reported that she could feel thecharacteristic “tingling” sensation in her lip that usually preceded theeruption of a “fever blister”. Upon feeling the “tingling”, the subjectmade one application of a 10% solution of the subject topical antiviral.Within 12 hours, the tingling sensation ceased and there was no eruptionof a blister.

EXAMPLE 19 Treatment of Herpes

A 47 year old Caucasian female with a history of frequent herpes simplexeruptions she characterized as “cold sores” or “fever blisters”. Thesubject presented with a rather large, painful blister on her upper lip.The subject applied a 10% aqueous solution of the subject antiviral.Within 12 hours there was a decrease in pain and soreness and she beganto experience a drying of the lesion she described as “a sort ofscabbing”. She made a second application approximately 12 hours later.The lesions continued to resolve. At approximately 36 to 48 hour afterthe initial application the lesion was described as “pretty well gone”.

EXAMPLE 20 Treatment of Herpes

The subject was a 17 year old Caucasian male who presented with numerouspainful white sores in his mouth and throat areas typically described as“stomach sores” or herpes. The subject suffered from the lesions forapproximately two days. He could barely eat solid food due to thediscomfort and pain. Before bedtime on day two, the subject tookapproximately ½ ounce of a 10% aqueous solution of the subject antiviraland swished it around in his mouth and spit it out. Upon awakening,approximately 8 hours later, the subject reported his mouth did nothurt, but that the sores were still there. He applied a second dose in asimilar manner that morning. That evening he reported that he could eatwithout pain, but felt that one or two “spots” were still tender. Hemade no more applications. At approximately 24 hours from the firstapplication, the subject reported that his mouth was healed.

EXAMPLE 21 Use Of Picolinic Acid For Orocutaneous Herpes Simplex

Data on over 60 patients who have used a picolinic acid ointment appliedto orocutaneous herpetic lesions. The study was focused on the first 48hours following treatment of a herpes outbreak with 10% picolinic acidin an ointment (PA Ointment). All subjects in the test group whoexperienced the initiation of a herpes eruption were treated within 24hour with PA Ointment. Data collected over the first 8 days were: levelof pain and discomfort during the first 24 hours; inflammation at theeruption site within 24 hours; if advanced infection and blisters werepresent, collapse of blisters within 2 hours; perception of pain; andattenuation of infection within 48 hours were recorded.

All of the patients universally experienced resolution of pain,swelling, inflammation, and vesicle formation within 24 to 48 hours ofinitiating its use. In previous episodes, all of these patients hadfailed adequate symptomatic control of their viral eruptions withconventional therapy. There was no toxicity to the normal or viralinfected skin areas noted in this study.

EXAMPLE 22 Treatment of Chickenpox

The subject was a 10 year old female Caucasian presenting with typicalchickenpox. The subject developed the typical rash on her torso,particularly her back and abdomen. When the rash was still in its earlystages, before full-blown blistering occurred, groups of the lesionswere marked by encircling with an indelible marker. A 10% solution ofthe subject antiviral was applied to the lesions inside the markedareas. These lesions did not erupt into blisters and the subjectreported that the treated areas did not itch like the others.

EXAMPLE 23 Metastatic Colon Cancer PREPARATIONS CONTAINING METALCHELATING PICOLINIC ACID AND DERIVATIVES FOR THE TREATMENT ANDPREVENTION OF SPECIFIC DISEASE STATES EXAMPLE 1 Topical or IntravaginalPreparation of Picolinic Acid in a Absorption Base

A topical or intravaginal preparation of picolinic acid in an absorptionbase is made by incorporating 0.001% to 99.9%, preferably 1% to 50%,most preferably 5% to 20% picolinic acid into an absorption base. Anabsorption base generally is an anhydrous base which has the property ofabsorbing several times its weight of water to form an emulsion andstill retain an ointment-like consistency. Absorption bases may vary intheir composition but generally are a mixture of animal sterols withpetrolatum, such as Hydrophilic Petrolatum, U.S.P. The most commoncommercially available products are Eucerin and Aquaphor (Beiersdorf)and Polysorb (Fougera). One preferred embodiment of the topicalpreparation is made by dissolving 10% picolinic acid in deionized waterand then incorporating the solution into an equal amount of Aquaphor, ona wt/wt basis. Further, the picolinic acid or derivatives can beincorporated into a balm or stick for application to the lips to treatherpes infections. It will be appreciated that picolinic acidderivatives can be used in place of the picolinic acid in the topicalpreparation. It will be appreciated that an appropriate concentration ofa substituted picolinic acid derivative can be used in place of thepicolinic acid without departing from the scope of the invention. Itwill be appreciated that such preparations can be used to treat topicalconditions such as virus infections, fungal infections, susceptiblebacterial infections, radiation assault, including ultraviolet, medicalor atomic radiation, skin cancers or any other condition mediation bythe above described mechanisms.

EXAMPLE 2

Picolinic Acid Solution

Picolinic acid can be employed topically, for vaginal installation, forinhalation or as a mouthwash as a 0.001% to 99.9%, preferably 1% to 50%,most preferably 5% to 20% aqueous solution. One preferred embodiment ofthe solution is prepared by dissolving an appropriate amount ofpicolinic acid in an appropriate amount of deionized water to form a 10%solution. The preparation can be used in any pharmaceutically acceptablemanner including topically, orally, on the mucosa and so forth. It willbe noted that picolinic acid derivatives can be used in place of thepicolinic acid, if desired. For inhalation purposes, the solution may beatomized with the use of an appropriate device.

As stated above, it is likely that picolinic acid will interfere withthe replication of the retroviruses by chelating zinc and preventing theactivity of certain zinc finger proteins. Therefore, a suitablepreparation of a chelating material, for example, picolinic acid orderivative may be used for vaginal application to prevent infection withany virus containing zinc finger proteins as an essential component ofthe viral replicating machinery, i.e. transcription factors. Suchviruses include, but are not limited to, human papilloma viruses (E6 andE7 zinc finger proteins) and the AIDS virus (tat protein). As explainedabove, the picolinic acid and substituted derivatives thereof are usedto attack the p7 protein having two zinc finger segments which is foundin the HIV virus which causes AIDS, which is essential for packaging RNAin the viral particles.

The preparation may be prepared by incorporating approximately 5% to 20%picolinic acid in a suitable base, such as Aquaphor, and instilling theointment vaginally before coitus. It also may be possible to prepare adouche of approximately 0.001 % to 99.9%, preferably 1% to 50%, mostpreferably 5% to 20% picolinic acid in deionized water and used beforeand after coitus. Such preparations may be used prophylactically toprevent infection with these viruses.

Furthermore, the preparations may be used vaginally to treat the uterinecervix infected with papilloma virus.

A condom containing approximately 5% to 20% picolinic acid or derivativemay be used to prevent replication of the viruses in the vaginal andcervical cells in the event the condom fails or ruptures. It will beappreciated that an appropriate concentration of a substituted picolinicacid derivative can be used in place of the picolinic acid withoutdeparting from the scope of the invention.

EXAMPLE 3 Ocular Preparation

A preparation of picolinic acid or a derivative thereof can be preparedfor the treatment of ocular herpes or other retroviral infections of theeyes. The topical or intraocular ophthalmological preparation includesapproximately 0.01% to approximately 5% picolinic acid or one of itssubstituted derivatives in an appropriate, ion-free vehicle, such asmethylcellulose. The preferred embodiment would include 0.01% ofpicolinic acid or derivative for topical ophthalmological application.However, the invention is intended to include a broader range ofconcentrations of picolinic acid or derivative thereof. It will beappreciated that an appropriate concentration of a substituted picolinicacid derivative can be used in place of the picolinic acid withoutdeparting from the scope of the invention.

EXAMPLE 4 Inhalation or Intranasal Formulation

A product suitable for intranasal administration for treatment of upperrespiratory diseases includes approximately 3 mM picolinic acid in asuitable isotonic vehicle. One example is 3 mM picolinic acid in Ocean®Nasal Mist (Fleming & Co., St. Louis, Mo.). The intranasal solution in arange between 0.01 mM to 50 mM, preferably 0.1 mM up to 20 mM picolinicacid or greater.

Likewise, a solution for pulmonary inhalation is prepared by addingpicolinic acid to normal saline for nebulization, the resulting solutionbeing in a range of 0.001% to 50% picolinic acid, derivative or analogin saline or sterile distilled water for nebulization.

EXAMPLE 5 Systemic Administration

A systemic preparation of a picolinic acid, its derivatives or analogscontaining approximately 1% to 100% active ingredient may beadministered orally, intravenously or by any acceptable route for thetreatment of cancer and systemic infections. For example, picolinic acidprepared in 00 gelatin capsules at 500 mg per capsule has been shown tobe effective in the control of metastatic cancer. The preparation can beprovided as a flavored oral solution. Likewise, an injectable form maybe prepared.

As set out above, the safe and effective daily systemic dose may rangefor 250 mg to 140 grams for a 70 Kg subject, with the preferred rangebeing 250 mg to 6 grams, and the most preferred dose being 500 mg to2000 mg.

EXAMPLE 6 Gastric or Peritoneal Lavage

A solution of up to 99%, preferably about 20% active ingredient, forexample picolinic or fusaric acid, can be used for gastric andperitoneal lavage for the treatment or control of infections or cancer.

Heat Shock Proteins, Viral Infection and Inflamatory Response

Prokaryotes and eukaryotes express numerous heat shock proteins (Hsps)in response to stress, including heat shock, exposure to heavy metals,hormones and viral infections. These heat shock proteins mediate andexacerbate the inflammatory response. Furthermore, Hsp27, the mostcommon hsp found in mammals and has been shown to be involved in cancer,such as breast cancer. An increase in cellular levels of Hsp27 and Hsp70increases the resistance of cancer cells to apotosis.

The stress response which includes numerous forms of physiological andpathological stresses is involved in viral infection also. A prominentfeature of this response is the synthesis of a discrete set of proteins,known as the heat shock proteins which, at present, are denotedmolecular chaperones. The role of these proteins are illustratedschematically in FIG. 13. During infection by certain viruses, heatshock proteins act as intracellular detectors that recognize malfoldedproteins. In general aberrant protein folding and degradation reactionscaused by exogenous or endogenous factors have emerged as a cause ofcancer and aging. Researchers have found that viruses are able toactivate heat shock proteins. For example, Hsp70 (DnaK) is induced byadenovirus, herpes virus, cytomegalovirus, and other viruses. DnaJproteins are zinc finger proteins, defined by the J domain, which isessential for stimulation of the Hsp70 (DnaK) ATPase activity. Thus, theresults suggest that there may be a relationship between the stressresponse and the cytopathic effects of certain viruses such as herpesviruses. Hsp70 has a protective role in inflammation, infection and aregulatory role in cytokine biosynthesis. Hsp70 exists in cells inequilibrium between its free state, in the cytoplasm, and its boundstate, protecting proteins in the nucleolus, interacting with ribosomalproteins to either refold some of the unfolded ribosomal proteins or bysolublizing the denatured ribosomal proteins to facilitate their use andincrease turnover rate. During release from heat shock and as thenucleolus begins to recover its normal activities,. a significantportion of Hsp70 returns to the cytoplasm. The inventor believes thisprotein—protein interaction has profound implications for viralreplication, since viruses control ribosomal protein synthesis andduring such process many of them damage the cells. Hence cellularinflammatory responses to viral infection are part of the organismdefense against viruses. The compounds and methods of the presentinvention can be used to block the action of the DnaJ zinc fingerproteins when excessively expressed. By blocking the DnaJ zinc fingerproteins, which are essential for stimulation of the Hsp70 (DnaK) ATPaseactivity, the Hsp70 destroying activity of Hsp70 ATPase enzyme will beinhibited, thus blocking the effect of the Hsp70. This blocking of theDnaJ zinc finger protein which is required for the enzyme activity,therefore, will reduce the stress reaction in virally infected cells.

Furthermore, it is believed that the response of cells to stress, suchas exposure to UV radiation, chemicals, bacteria, parasites or fungi isassociated with the induction of heat shock proteins. Formulations of1%, 5% or 10% picolinic acid in an appropriate vehicle, such as anabsorption base, have been used to treat sunburn in over 25 cases.Hence, the compounds and methods of the present invention can be used toblock inappropriate and excessive cellular stress response caused, inthis example, by UV radiation. Moreover, the inflammation associatedwith acne has been treated in more than 35 cases, in both adults andteenagers, with a 5% to 10% aqueous solution of picolinic acid. Asdetermined by computer modeling, the compound binds with DnaJ andreduces or eliminates the effect of the DnaJ blocking zinc fingerproteins inactivating enzymes and hence controls the inflammatoryresponse induced by bacteria in common acne.

Presently recognized disease states involving inflammatory responsesthat may be susceptible to the inventor's methods also includearthritis, Alzheimer's disease. Furthermore, Arterosclerosis has aninflammatory and proliferative component which may be blocked by theinstant compositions and methods. By way of further example, it is knownthat inflammatory cytokines play some role in Alzheimer's disease.Inflammatory molecules and mechanisms are present in the disease andinflammation may be an essential component of the disease sufficient tocause neurodegeneration. Thus, suitable doses of the disclosed picolinicacid derivatives, particularly those shown to cross the blood-brainbarrier, can be used to treat the inflammatory component of the disease.

Furthermore, the claimed invention is intended to apply to otherpathological conditions which involved inflammatory responses bothpresently known and unknown. Another example of a salutory use of theclaimed invention is to prevent fibroblast production in the eye aftercataract surgery and the implantation of artificial lenses, particularlythose made from polymeric materials. The lenses, due to surfacecontamination, induce proliferation of fibroblasts with resultantopacity of the lens. Presently, 5-fluorouracil is instilled in the eyeto control fibroblast proliferation. However, the 5-fluorouracil istoxic and not without untoward effects. Topical application of thechelating agents of the present invention, specifically as discussedbelow, can control fibroblast proliferation. The combination of theantifibroblast activity and the antiangiogenesis activity make chelatingagents, such as fusaric acid, ideal drugs after ophthalmologic surgery.

It will be appreciated by those skilled in the art that the inventor hasdisclosed the best mode by which he presently understands picolinic acidand its derivatives, to function in controlling inflammatory response.However, the scope of the appended claims is intended to include othermechanisms of action, both presently known and unknown, which includemetal ion containing proteins as mediators in the inflammatory responseincluding, but not limited to, parasitic diseases such as toxoplasmosisand malaria.

Virus and Cancer Vaccine

It is clearly established that zinc finger proteins of viruses areessential for viral replication and that mutations of the zinc fingerdomain produce non-infective viral particles. Thus, the zinc fingerdomain is an essential component of the virus, without which it cannotreplicate, exit from the cells and infect other cells. Moreover, sincethe zinc finger domain is highly conserved, it generally does notmutate. However, when and if the zinc finger domain does mutate, themutation is lethal, that is, the virus dies.

Based upon the foregoing understanding of the function and activity ofmetal ion containing proteins (metalloproteins), the inventor hasdiscovered a method of preparing vaccines using metalloproteinsessential for virus replication and packaging of viruses. In general, avirus is treated with the metal chelating agent, picolinic acid or oneof the afore described derivatives or other suitable derivatives, forexample, which denatures the zinc finger proteins of the virus. Thepicolinic acid, for example, distorts the protein configuration to suchan extent that it becomes immunogenic and new antigenic sites areexposed. Thus, the patient develops antibodies against viral replicasesand viral proteins involved in packaging the viruses.

Generally speaking, the method includes an antigenic approach to virusand cancer treatment using zinc finger protein-antigen complexes derivedfrom an individual patient's tumors or virus-containing tissues as wellas zinc finger protein-antigen complexes derived from non-autologoustissue sources. The resulting vaccines are used to stimulate a cellularimmune response against viruses, primary tumor cells as well asmetastasis.

More specifically, in the method of the present invention the DNA or RNAis removed by standard nucleic acid technology and discarded. Afterremoval of the nucleic acid, the viral metalloproteins are mixed withthe picolinic acid or a derivative thereof in an emulsion to preventrenaturation of proteins by binding to metal ions. The zinc fingerproteins are captured by beads derivatized with an analog of picolinicacid containing a side chain at the 5 position, or other suitableposition, of sufficient length to allow binding of the protein to thepicolinic acid metal ion. The zinc finger proteins then are conjugatedto an immunogenic protein such as keyhole limpet hemocyanin (KLH), forexample as taught in U.S. Pat. No. 5,243,041 (Re. 35,583). The finalantigen, consisting of the KLH-zinc finger protein-picolinic acidderivative. The picolinic acid derivative is covalently bonded to thezinc finger protein to prevent renaturization of the zinc fingerprotein. The proteins are injected into animals following standardschedules.

Vaccines against certain viruses, particularly against HIV are difficultto produce. The ineffective nature of the hosts natural immune responseindicates that a vaccine effective against HIV must provide highlyspecific protective immunity and, more important, must providesterilizing immunity. This problem is compounded by the large number ofHIV-1 strains. In fact, HIV-1 can be considered a quasi-virus thatmutates continuously and thus vaccines for one strain are notcross-reactive or only minimally neutralizing for different strains,even in the same patient. However, the present method of preparing avaccine using a highly conserved zinc finger protein derived from HIVwill have cross antigenicity and will overcome problems associated withknown methods of vaccinating against HIV.

It will be appreciated also that metalloproteins have been shown to playan important role in cancer. Metalloproteins, such as MPS-1 have growthstimulating functions that may be implicated in cancer cellproliferation and in metastasis. Thus, a vaccine, prepared as describedabove with reference to viral metalloproteins can be used to immunizepatients against the deleterious effects of zinc finger proteins orother metalloproteins that play a role in cancer cell growth andproliferation.

It will be appreciated that various changes and modifications may bemade in the preparations and methods described and illustrated withoutdeparting from the scope of the appended claims. It will be appreciatedthat the description of the specific embodiments of the preparation fortopical use is intended to include pharmacologically acceptedconcentrations of the above described substituted derivatives ofpicolinic acid, as well as the picolinic acid itself. Oral or injectableforms of the preparations also are contemplated by the invention.Further, suitable preparations, other than topical preparations, ofmetal chelating compounds may be employed for the treatment ofadenocarcinomas and squamous cell carcinomas. The preparation may beused alone or in combination with other chemotherapeutic agents. Thepicolinic acid or derivative can be included with various chemical ormechanical carriers, both known and heretofore unknown, to allowpenetration or entry into tumors. Furthermore, the preparations may beused to treat a wide spectrum of proliferative and viral diseasesmediated by zinc finger proteins, zinc ring proteins or other metal iondependent proteins or enzymes. Therefore, the foregoing specificationand accompanying drawings are intended to be illustrative only andshould not be view in a limiting sense.

Zinc-Finger and Iron-Finger Hormone Receptor Proteins and Aging andCarcinogenesis

At physiological concentrations, transition metal ions, such as iron,cobalt and copper are essential elements for biological functions; athigher levels, however, they are toxic. This is particularly true foriron. Toxicity of the transition metal ions, particularly iron, is dueto the fact that protein domains are present within key enzyme andtranscriptional regulatory molecules (DNA-binding proteins) whichnormally bind zinc (zinc finger domains) but which can substitute zincby other transition metals that are present in the cell. Elevated levelsof iron contribute to carcinogenesis in several ways; iron has thecapacity to generate highly reactive free radicals that damage DNA, andrapidly proliferating transformed cells have increased requirement foriron for DNA replication (ribonucleotide reductase) and for energyproduction by mitochondria.

Iron can replace zinc in the zinc-containing hormone-receptor proteinsfor testosterone, progesterone and other hormones. Iron may alsogenerate free radicals which damage DNA in specific regulatory regionsand potentially induce carcinogenesis in the prostate, uterus, and otherorgans. Thus, classical hormones can modulate iron-finger receptorproteins. The hormones potentiate the destructive actions of freeradicals, mediated by abnormal iron-finger receptor proteins, onregulatory regions of DNA. The inventor determined that it is feasibleto maintain zinc-finger proteins in an undamaged zinc-containing form byusing a combination of specific agents, such as iron chelators andradical scavengers, respectively, interfere with the formation of bothaberrant iron-finger proteins and free radicals. Thus, picolinic acid,fusaric acid, and pharmacologically acceptable derivatives thereof, inthe dosages discussed above, as well as chelators yet unknown, can beused to prevent the formation of aberrant iron-finger proteins involvedin carcinogenesis and aging. Free radical scavengers include knownanti-oxidants such as vitamin E and so forth.

Antiangiogenesis

Angiogenesis is defined as the development of blood vessels. It recentlyhas been understood that one method for controlling the growth of tumorsis to control angiogenesis related to the tumor. Simply put, bycontrolling or preventing angiogenesis, one can prevent blood flow tothe tumor resulting in tumor death.

Furthermore, there are other disease states in which angiogenesiscontributes to disease progression or symptoms. For example,angiogenesis has been implicated in retinopathies, such as diabeticretinopathy, which can result in blindness. Likewise, there can beincrease angiogenesis and untoward fibroblast production afterophthalmologic surgery that can result in vision impairment andblindness. Hence, the control or retardation of blood vessel developmentcan control or halt tumor growth and result in tumor death by deprivingthe tumor cells of necessary blood flow and nutrients. Likewise,controlling the proliferation of blood vessels in the eye can alleviateretinopathies, including diabetic retinopathy and also preventpost-surgical complications.

The instant invention is effective in controlling undesirableangiogenesis. For example, picolinic acid, or derivatives thereof,chelate copper, zinc and iron. Copper, zinc and iron are obligatorycofactors of angiogenesis. They stimulate endothelial cell migration andproliferation and activate vascular growth factors. Moreover, they areessential for collagen synthesis. The novel chelating agents of thepresent invention chelate copper, zinc and iron and thereby inactiveribonucleotide reductase, zinc finger proteins and other coppercontaining proteins essential for cell growth. Furthermore, thechelating agents inhibit collagen cross-linking by interfering withprolyl and lysil hydroxylases.

The inventor has determined antiangiogenic effect can be achieved with apharmacologically acceptable dose of chelating agent, includingpicolinic acid or derivatives thereof. The effective dose of picolinicacid is wide ranging, depending upon clinical response, and can rangeanywhere from 100 mg to 8 grams daily, preferably 250 mg to 6 gramsdaily, most preferably from 500 mg to 4 grams daily. The dose can beadministered by any acceptable route including systemically, orally ortopical in an ophthalmic preparation. An illustrative systemic dosageregimen would provide for 500 mg picolinic acid given orally over a five(5) week escalation from approximately 500 mg picolinic acid daily up to4 grams daily. The patient also should maintain a diet low in transitionmetal ions and drink only deionized water. The primary end-point of thetreatment, in cases of cancer, is time of survival. Secondary end-pointsare reduction of tumor volume and reduction of or delay in progression.It also will be appreciated by those skilled in the art that theprophylactic administration of the novel chelating therapy will preventthe development of the cancers. Thus, the antiangenogenic therapy withthe novel invention in cancers is dependent on stage of cancer and tumorsize, i.e. prevention for microscopic disease, intervention for smalltumors, and adjuvant therapy for large, end-stage tumors.

Furthermore, it will be appreciated that the novel therapy of thepresent invention can be used to augment other prior art or conventionalcancer treatments, such as chemotherapy and radiation therapy. Theinvention has direct cytotoxic effects, as stated above. Furthermore,the antiangiogenic effect enhances its effectiveness. Moreover, addingthe novel therapy to a conventional chemotherapy regimen allows for thereduction of dose of the conventional chemotherapeutic agents andprevents multidrug resistance from developing in the patient. Drugresistance to the chelating agent is unlikely because blood vesselendothelial cells are a genonically stable, diploid population.

In the treatment of ophthalmologic diseases, the end-point is reductionin vessels proliferation and malformation in the retina and improvedprognosis. Likewise, it will be appreciated by those skilled in the artthat the prophylactic administration of the novel chelating therapy willprevent the development of the retinopathies. Oral or other systemicdosing of approximately 250 mg to less than 140 gram per day, preferably250 mg to 2000 mg per day is optimal. An ophthalmic preparation, i.e.drop or ointment, containing approximately 0.01% to 99% activeingredient, most preferably 5% to 10% active ingredient can beadministered to the eye one to four times daily.

Various changes and modifications may be made in the foregoing inventionwithout departing from the scope of the appended claims. Therefore, thespecification and accompanying drawings should be considered asillustrative of the best mode of working the invention, but should notbe construed in a limiting sense.

What is claimed is:
 1. A method of treating retinopathy in the retina ofthe eye of an individual comprising the administration of at least onechelating agent to the individual to retard the development of bloodvessels in the retina of the eye, wherein said agent is represented bythe following structure:

or a pharmacologically acceptable salt thereof, wherein R₁, R₂, R₃ andR₄ are selected from the group consisting of a carboxyl group, methylgroup, ethyl group, propyl group, isopropyl group, butyl group, isobutylgroup, secondary butyl group, tertiary butyl group, pentyl group,isopentyl group, neopentyl group, fluorine, chlorine, bromine, iodine,and hydrogen.
 2. The method of claim 1 wherein the retinopathy isdiabetic retinopathy.
 3. The method of claim 1 wherein the retinopathyis a post-surgical retinopathy.